Project Summary: How chromatin domains are established and maintained in higher eukaryotes are poorly understood, especially during development. Covalent modifications of histones carry significant amount of information that complement to genetic information in dictating developmental process. Mutations in histone modifying enzymes are commonly found in human diseases, further underlying their fundamental roles in cells. One such enzyme is MLL1 (mixed lineage leukemia), which is evolutionarily conserved and promotes 5' Hox gene transcription. MLL1 is a histone methyltransferase for H3 lysine 4, a mark highly enriched at gene promoters and enhancers. Studies have found that MLL1 plays important roles in normal and malignant hematopoiesis. Knockout MLL1 gene leads to profound defects in hematopoietic stem cells compartment and blocks MLL1 fusion protein mediated leukemogenesis. Despite these findings, the obligatory function of H3 K4 methylation in MLL1 dependent biological pathways remains unclear. Our biochemical studies have revealed that the MLL1 methyltransferase activity is tightly regulated by three proteins including WDR5, RbBP5 and ASH2L. We, and others, further demonstrate that MLL1-WDR5 interaction is essential for complex integrity. Blocking this interaction abolishes MLL1 activity. Based on these results, we have developed a highly specific chemical probe that targets MLL1 methyltransferase activity by disrupting MLL1 complex assembly. Using this novel probe, we will directly address the function of MLL1 mediated H3 K4 methylation in malignant process (without affecting other MLL1 functions). This novel probe also, for the first time, allows us to examine the dynamic and reversible functions of H3K4 methylation during normal cell differentiation. Our studies will have high impact in the chromatin field and translational research.